ABSTRACT Drugging Gram-negative bacteria in the clinic is an urgent unmet need due to rapidly-evolving resistant strains, the inability of conventional antibiotics to penetrate the outer cell wall, and off-target in vivo drug toxicities. Antimicrobial peptides (AMPs) and other small molecule antibacterial leads have shown promise in preclinical testing for killing multi-drug resistant Gram-negative pathogens, but have faced significant challenges in clinical translation as a result of inferior therapeutic outcomes in vivo. This proposal addresses the shortage of novel treatment strategies for multi-drug resistant Gram-negative pathogens by exploiting the pathogen's cell surface glycans and local environment to deliver antimicrobial payloads. Long-circulating, pro-drug constructs will be engineered that selectively target the site of infection after systemic administration and activate in response to proteolytic activity specific to the infected tissue microenvironment. The proposed antimicrobial agents, termed antimicrobial conjugates (AMCs) consist of a pathogen-specific targeting agent, a microenvironment-specific cleavable linker, and a bactericidal payload. This modular design allows the exploration of different components to optimize the conjugate's activity. The proposed AMCs will be designed and extensively evaluated in vitro and in animal models for toxicity and antimicrobial activity by a joint team at MIT composed of Drs. Sangeeta Bhatia, Timothy Lu, Laura Kiessling, and Bradley Pentelute. Their labs will leverage expertise with protease-responsive nanomaterials, synthetic biology and computational design, protein-glycan recognition processes, and bioconjugation and rapid peptide synthesis technologies, respectively, to advance the development of AMCs. This new therapeutic modality has several advantages: the high level of specificity for pathogen targets will limit toxicity to host, enabling the use of less selective antimicrobial agents, the conjugates will have increased pharmacokinetics, and the narrow spectrum activity will avoid the spread of general resistance mechanisms between species and limit damage to the host microbiota. Completion of the project will generate lead antimicrobial conjugates for the treatment of resistant Gram-negative infections. Collectively optimizing the therapeutic profiles of lead compounds will identify top candidates that can be advanced for pre-clinical trials, with the potential to deliver a therapeutic strategy that effectively bypasses acquired Gram-negative antibiotic resistance.